Cytotoxic drugs are now used at some time during the course of the treatment of most cancer patients. Cytotoxic drugs can cure some primary and metastatic cancers and be effective in decreasing tumor volume, treating symptoms and even prolonging life in many types of cancers. The starting dose of the cytotoxic drug in cancer patients is based on preclinical evaluation of the therapeutic dose in different tumor cell lines in vitro and in animal tumor modes in vivo as required by the Guidelines from the Food & Drug Administration for Nonclinical Evaluation for Anticancer Pharmaceuticals (ICH S9). In experimental xenograft tumor models the dose-response curve is usually steep in the linear phase, and a reduction in dose when the tumor is in the linear phase of the dose-response curve, almost always results in a loss in the capacity to cure the tumor effectively before a reduction in the antitumor activity is observed. Although xenograft models may not represent the ideal model for human malignancies, the general principles have been applied to the clinical setting. Such empirical modifications in dose represent a major reason for treatment failure in patients with drug-sensitive tumors who are receiving chemotherapy in either the adjuvant or advanced disease setting.
Another reason for treatment failure in patients with drug-sensitive tumors who are receiving cytotoxic drug is the effect of the cytotoxic drug on the microenvironment of the tumor since the chemotherapy may cause mechanisms that can interfere with its antitumor activity on the tumor itself. The present invention provides a method to include CTO to modulate early and late changes induced by the cytotoxic drug in the microenvironment to minimize the interference with its antitumor activity to improve the sensitivity of the chemotherapy to achieve treatment success. More particularly, CTO is selected as the combinatorial targeted drug to control several of the mechanisms induced in the tumor microenvironment that interfere with the sensitivity and efficacy of the cytotoxic drug.
Since the development of chemotherapy in the 1950s and 1960s that resulted in curative therapeutic strategies for patients with several types of solid tumors and hematologic malignancies the understanding of genetic changes that can result in drug resistance has provided innovative therapeutic strategies; but unfortunately, these studies have not heretofore, considered the early effect of the chemotherapy on the microenvironment of the tumor in the determination of the sensitivity and effective doses required to achieve treatment success. The present invention provides a method for i) evaluating the effect of a cytotoxic drug on the microenvironment of a specific tumor type, ii) identifying the mechanisms that mediate the anticancer activity or that interfere with the anticancer activity, iii) selecting a combinatorial drug, for example CTO, that minimizes or prevents the mechanisms interfering with the anticancer activity, iv) determining the pharmacodynamic interaction between the cytotoxic drugs and the combinatorial chemotherapy with CTO, and v) determining an optimum combination of the cytotoxic drug and CTO to achieve maximum efficacy and least toxicity.
In other words, the present invention provides a method to determine the effective dose of the cytotoxic chemotherapy that is based on enhancing its sensitivity by combining it with carboxyamidotriazole orotate (CTO), an orotate salt of carboxyamidotriazole (CAI). CAI is an inhibitor of receptor-operated calcium channel-mediated calcium influx, and is shown to have antiproliferative and anti-invasive functions in several human cancer cell lines, including human glioblastoma cells (Ge et al, 2000). By interrupting calcium mobilization as a second messenger, CAI can inhibit calcium-sensitive signal transduction pathways, including the release of arachidonic acid and its metabolites; nitric oxide release; the generation of inositol phosphates; and tyrosine phosphorylation (Ge et al, 2000; Kohn et al, 1992). CAI inhibits phosphorylation of cellular proteins STATS and CrkL, and induces apoptosis in imatinib mesylate-resistant chronic myeloid leukemia cells by down-regulating bcr-abl (Alessandro et al, 2008). CTO targets the tumors as well as the microenvironment of the tumor and mechanisms that may induce drug resistance or interfere with the antitumor activity.
The timing and duration of the combination therapy may be determined to be from the start of the cytotoxic drug chemotherapy or at various stages during the therapeutic regimen based on the understanding of the dynamics and extent of the effect of the cytotoxic chemotherapy on the tumor microenvironment. Current principles guiding the selection of chemotherapeutic cytotoxic drugs and of their doses do not consider their impact on the tumor microenvironment. As a result the dose selected to treat a cancer may include an extra amount to overcome early interference in the microenvironment and thus may require reevaluation to achieve optimum treatment success.
Currently, the established dosing regimens for cytotoxic drugs have not factored in this interference in sensitivity of the cytotoxic drugs, hence leading to increase in the dose used. Dose-dense strategies are used to achieve tumor shrinkage but these cause severe toxicities in cancer patients and adversely affect their quality of life. There is a critical need to prevent or reduce this interference in sensitivity instead of using dose dense regimens. The present invention provides a paradigm for the development of new drug therapeutic programs using existing drugs in use or new drugs that design combinations of cytotoxic drugs and CTO where applicable to maximize the sensitivity of the drugs to achieve treatment success rather than taking a does dense approach.
Chemotherapy with cytotoxic drugs is presently used in four main clinical settings: 1) primary induction treatment for advanced disease or for cancers for which there are no other effective treatment approaches, 2) neoadjuvant treatment for patients who present with localized disease for whom local forms of therapy such as surgery and/or radiation are inadequate by themselves, 3) adjuvant treatment to local methods of treatment, including surgery and/or radiation therapy, and 4) direct instillation into sanctuary sites or by site-directed perfusion of specific regions of the body directly affected by the cancer. Physicians” Cancer Chemotherapy Drug Manual, ed, E. Chu, V. T. DeVita, Jr, 2010. The present invention provides a paradigm for the development of new drug therapeutic programs in each of the above four clinical settings to increase the sensitivity of some cytotoxic drugs.
According to the method of the invention, it is 1) necessary to identify whether a tumor is responsive to a specific cytotoxic drug, 2) to identify a profile of molecular targets in the tumor microenvironment that may potentially interfere with its anticancer activity, and 3) to select the most suitable combinatorial regimen of the cytotoxic drug and targeted chemotherapy, for example a cytotoxic drug and CTO. Among the problems currently associated with the use of cytotoxic drugs to treat cancers are the high doses required, toxicity towards normal cells, lack of selectivity and sensitivity, immunosuppression and drug resistance because molecular targets that interfere with the anticancer activity are not controlled.
Drug resistance may also be caused by malignant cells becoming resistant to the drug and a number of cellular mechanisms are probably involved in altering metabolism of the drug, permeability of the cells to the drug or accelerated elimination of the drug, altered specificity of the inhibited enzyme, or amplification of certain genes involved in resistance to chemotherapy or biologic therapy. This is observed after multiple exposures to the drug as described below.
For example, amplification of the gene encoding dihydrofolate reductase is related to resistance to methotrexate, while amplification of the gene encoding thymidylate synthase is related to resistance to treatment with 5-fluoropyridines.
The therapeutic benefit of temozolomide depends on its ability to alkylate/methylate DNA, which most often occurs at the N-7 or O-6 positions of guanine residues. This methylation damages the DNA and triggers the death of tumor cells. However, some tumor cells are able to repair this type of DNA damage, and therefore diminish the therapeutic efficacy of temozolomide by expressing O-6-methyguanine-DNA methytransferase(MSMT) or O-6-alkyguanine—DNA alkyltransferase (AGT or AGAT). In some tumors epigenetic silencing of the MGMT/AGT gene prevents the synthesis of this enzyme, and as a consequence such tumors are sensitive to killing by temozolomide. Conversely, the presence of MGMT protein in brain tumors predicts poor response to temozolomide and these patients receive little benefit. But resistance to temozolomide is related to other factors as well. In GBM patients the tumor responds to temozolomide at first but later, even with increased doses, the tumor becomes refractory after a few courses. This suggests that other interfering factors may be reducing the sensitivity of temozolomide to cancer cells.
Therefore, there is need to understand the mechanism of resistance to temozolomide that may be related to other mechanisms since it is one of the few drugs that crosses the blood brain barrier and used in treatment of malignant gliomas and glioblastoma multiforme, but for the drug resistance that develops after a few courses.
There is some evidence that changes in the tumor microenvironment induced by doxorubicin may impede its delivery to the tumor target and therefore a combinatorial regimen of doxorubicin and CTO may provide a solution to maintain doxorubicin's sensitivity.
In some cases resistance to a drug may be linked to increased production of molecules (e.g., cytokines, calcium channel signaling, molecular signaling) in the tumor microenvironment that interfere with the sensitivity and efficacy of the cytotoxic drugs. Therefore, even the most rationally conceived drug molecule may fail because of mutational changes downstream from its intended target or metabolic features of tumors that never allow the drug to reach its target or that trigger feedback mechanism against the drug molecule.
Thus a rational approach to cancer drug therapy and development is needed that relies on the empirical evidence of tumor shrinkage with cytotoxic drugs, understanding mechanisms of action of the chemotherapy in the tumor microenvironment in real time, defining new lead structures directed to biochemical and molecular targets and causing the cytotoxic drugs to perform optimally. The present invention satisfies this need and provides additional advantages as well. The present invention is distinguishable from the prior art because none of the prior art addresses the issue of preventing and/or reducing the impact of cytotoxic drugs in the tumor microenvironment which interferes with its sensitivity in early stages of therapy before the more permanent biochemical changes lead to the drug resistance in later stages of therapy. The present invention provides a timely combinatorial therapy with CTO that targets the early interference and thus maintains or enhances the sensitivity of the cytotoxic drugs, thus avoiding the need for increase the dose to obtaining optimum efficacy.
The combination of a cytotoxic drug that induces some of the molecular targets controlled by CTO and an appropriate combinatorial dose and regimen of CTO thus provides more effective and less toxic paradigm for new successful cancer treatment programs, a fundamental object of the invention. The pertinent subject matter of the above references is specifically incorporated herein by reference.